Many modalities of electron microscopy (EM) can probe cellular structure at the nanometer scale. However, despite considerable progress over the past decade in developing high-resolution three-dimensional (3D) imaging, there remain important limitations reflecting an inherent trade-off between resolution and the size of the 3D volume. For demanding applications such as tracing neuronal processes, high resolution in the z axis, parallel to the direction of the electron beam of the electron microscope, in addition to the xy plane, is critical. Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) offers this capability, with xy and z resolution all <10 nm. FIB-SEM can generate 3D images with superior z-axis resolution, yielding data with isotropic voxels that is therefore more readily interpretable than available with other techniques.
However, previous FIB-SEM approaches have been severely limited in the volumes that they could image, with typical volumes being less than the extent of a single neuron. For example, obstacles blocking wider adoption of FIB-SEM include slow imaging speed and lack of long-term system stability, which caps the maximum possible acquisition volume, dictated by the limited imaging speed and the limited duration of smooth and consistent ablation. Because the FIB process is destructive, there is little room for error in the ablation-imaging cycle, which requires virtually perfect continuity and consistency.
Thus, what is desired are techniques that accelerate image acquisition while also improving FIB-SEM reliability, allowing a FIB-SEM system to operate continuously for long time periods while generating large imaged volumes.